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The aim of the study was to assess the presence of genes in ESBL-producing E. coli (ESBL-Ec) isolated from retail raw food in Nha Trang, Vietnam. A total of 452 food samples comprising chicken (n = 116), pork (n = 112), fish (n = 112) and shrimp (n = 112) collected between 2015 and 2017 were examined for the prevalence of ESBL-Ec. ESBL-Ec were detected in 46.0% (208/452) of retail food samples, particularly in 66.4% (77/116), 55.4% (62/112), 42.0% (47/112) 19.6% (22/112) of chicken, pork, fish and shrimp, respectively. Sixty-five out of the 208 (31.3%) ESBL-Ec isolates were positive for mcr genes including mcr-1, mcr-3 and both mcr-1 and mcr-3 genes in 56/208 (26.9%), 1/208 (0.5%) and 8/208 (3.9%) isolates, respectively. Particularly, there was higher prevalence of mcr-1 in ESBL-Ec isolates from chicken (53.2%, 41/77) in comparison to shrimp (22.7%, 5/22), pork (11.3%, 7/62) and fish (6.4%, 3/47). mcr-3 gene was detected in co-existence with mcr-1 in ESBL-Ec isolates from shrimp (9.1%, 2/22), pork (8.1%, 5/62) and fish (2.1%, 1/47) but not chicken. The 65 mcr-positive ESBL-Ec (mcr-ESBL-Ec) were colistin-resistant with the MICs of 4-8 μg/mL. All mcr-3 gene-positive isolates belonged to group A, whereas phylogenetic group distribution of isolates harboring only mcr-1 was B1 (44.6%), A (28.6%) and D (26.8%). PFGE analysis showed diverse genotypes, although some isolates demonstrated nearly clonal relationships. S1-PFGE and Southern hybridization illustrated that the mcr-1 and mcr-3 genes were located either on chromosomes or on plasmids. However, the types of mcr genes were harbored on different plasmids with varied sizes of 30-390 kb. Besides, the ESBL genes of CTX-M-1 or CTX-M-9 were also detected to be located on plasmids. Noteworthy, co-location of CTX-M-1 with mcr-1 or mcr-3 genes on the same plasmid was identified. The conjugation experiment indicated that the mcr-1 or mcr-3 was horizontally transferable. All mcr-ESBL-Ec isolates were multidrug resistance (resistance to ≥3 antimicrobial classes). Moreover, β-Lactamase-encoding genes of the CTX-M-1 (78.5%), CTX-M-9 (21.5%), TEM (61.5%) groups were found in mcr-ESBL-Ec. The astA gene was detected in 27 (41.5%) mcr-ESBL-Ec isolates demonstrating their potential virulence. In conclusion, mcr-1 and mcr-3 genes existed individually or concurrently in ESBL-Ec isolates recovered from retail raw food in Nha Trang city, which might further complicate the antimicrobial-resistant situation in Vietnam, and is a possible health risk for human., (Copyright © 2021 Elsevier B.V. All rights reserved.)